All animal work was approved by the University of Rochester University Committee on Animal Resources (UCAR) committee (UCAR 2007-065). Euthanasia method was sodium pentobarbital (100 mg per kg body weight) I.P. followed by cervical dislocation. Postinfection, the animals were observed closely and if any sign of discomfort such as unable to ambulate well enough to maintain hydration and caloric intake or greater than 10% weight loss is seen, the animal was euthanized immediately.
Bacterial Strain and Growth Condition
Bacterial strain used in this study was Salmonella typhimurium
wild-type strain ATCC14028s. Non-agitated microaerophilic bacterial cultures were prepared as previously described 
Human epithelial SKCO15 
, CaCo2BBE and HT29C19A cells 
were established cells lines derived from human colonic tumor cells. Cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), streptomycin-penicillin and L-glutamine. Monolayers of SKCO15, CaCo2BBE, and HT29C19A cells were grown on permeable supports (0.33 or 4.67 cm2
, 0.4 mm pore. Costar, Cambridge, MA, USA), as described in the previous publications 
Bacterial Colonization in the Polarized Epithelial Cells in vitro
Polarized human epithelial cells were colonized with equal numbers of the indicated bacteria for 30 minutes, washed with HBSS, and incubated in DMEM containing gentamicin (500 mg/ml) for the times indicated in our previous studies 
. The first 30-minute incubation allowed bacteria to contact the surface of the epithelial cells and inject the effectors in the host cells. After extensive HBSS washing, the extracellular bacteria were washed away. Incubation with gentamicin inhibited the growth of bacteria.
Streptomycin Pre-treated Mouse Model
Animal experiments were performed using specific-pathogen-free female C57BL/6 mice (Taconic, Hudson, NY, USA) that were 6–7 weeks old, as previously described 
. The protocol was approved by the University of Rochester University Committee on Animal Resources (UCAR). Water and food were withdrawn 4 hours before oral gavage with 7.5 mg/mouse of streptomycin (100 µl of sterile solution or 100 µl of sterile water in control). Afterwards, animals were supplied with water and food ad libitum. Twenty hours after streptomycin treatment, water and food were withdrawn again for 4 hours before the mice were infected with 1×107
CFU of S. typhimurium
(100 µl suspension in HBSS) or treated with sterile HBSS (control). Eight hours after infection, mice were sacrificed, and tissue samples from the intestinal tracts were removed for analysis.
Mouse Colonic Epithelial Cells
Mouse colonic epithelial cells were collected by scraping the mouse colon, including the proximal and distal regions. Cells were sonicated in lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA pH 8.0, 0.2 mM sodium orthovanadate, protease inhibitor cocktail). The protein concentration was measured using BioRad Reagent (BioRad, Hercules, CA, USA). Protein lysates collected from mouse colon were performed using a TritonX-100 buffer. We also measured the protein levels of claudins from both control and Salmonella infected colon as Triton soluble and insoluble fractions.
Mouse epithelial cells were scraped and lysed in lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA pH 8.0, 0.2 mM sodium orthovanadate, protease inhibitor cocktail), and then the protein concentration was measured. Cultured cells were rinsed twice in ice-cold HBSS, lysed in protein loading buffer (50 mM Tris, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol), and then sonicated. Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with primary antibodies. The following antibodies were used: anti-claudin-2, anti-claudin-3, anti-claudin-4, anti-claudin-7, anti-α-catenin, anti-VPS34 (Invitrogen, Grand Island, NY, USA), anti-p-SAPK/JNK, anti-SAPK/JNK, anti-p-c-jun, anti-p-c-jun, anti-p-EGFR, anti-EGFR, anti-AKT (Cell Signal, Beverly, MA, USA), anti-puma, anti-Villin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA.), or anti-β-actin (Sigma-Aldrich, Milwaukee, WI, USA.) antibodies and were visualized by ECL (Thermo Scientific, Rockford, IL, USA). Membranes that were probed with more than one antibody were stripped before re-probing.
Cultured epithelial cells HT29C19A were incubated with equal numbers of the indicated bacteria for 30 minutes and washed with HBSS. Immunofluorescent labeling of cells grown on inserts was performed as follows: cells were fixed for 10 minutes in 1% paraformaldehyde in HBSS and then washed in HBSS. Fixed samples were incubated in blocking solution (2% bovine serum albumin, 1% goat serum in HBSS) for 1 hour, followed by 4°C overnight incubation with primary antibodies. After a 60-minute incubation with secondary antibodies, the inserts were mounted with SlowFade (Invitrogen, Grand Island, NY, USA) followed by a coverslip, and the edges were sealed to prevent drying. Specimens were examined with a Zeiss LSM 710 Laser Scanning confocal microscope.
Colonic tissues from the proximal and distal portions of the colon were freshly isolated and embedded in paraffin wax after fixation with 10% neutral buffered formalin. After preparation of the slides as described above 
, slides were incubated in 3% H2
for 20 minutes at room temperature to block endogenous peroxidase activity, followed by incubation for 1 hour in blocking solution (2% bovine serum albumin, 1% goat serum in HBSS) to reduce nonspecific background. The samples were incubated overnight with primary antibodies at 4°C. Samples were then incubated with secondary antibodies for 1 hour at room temperature. Tissues were mounted with SlowFade. Specimens were examined with a Zeiss LSM 710 Laser Scanning confocal microscope.
Cells were grown as monolayers on collagen coated polycarbonate membrane Transwell supports (0.33 or 4.67 cm2, 0.4 mm pore. Costar, Cambridge, MA, USA). Cells were colonized with equal numbers of the indicated bacteria for 30 minutes, washed with HBSS, and incubated in DMEM containing gentamicin (500 ug/ml, Invitrogen Corporation) for the time indicated. Transepithelial resistance (TER) was measured with an epithelial voltohmmeter under open-circuit conditions (EVOM, World Percision Instruments, Sarasota, FL, USA). Each measurement was performed in triplicate.
Fluorescence Permeability in vivo
Streptomycin pre-treated mice were infected with bacterial strains for 24 hours. Fluorescein Dextran (Molecular weight 3000 Da, diluted in HBSS) was gavaged (50 mg/g mouse). Four hours later, mouse blood samples were collected by cardiac puncture. Fluorescence intensity of the plasma was measured on a fluorescent plate reader 
SKCO15 cells were grown as monolayers on collagen coated polycarbonate membrane Transwell supports. The cells were transfected with esiRNA human CLDN2 (Sigma-Aldrich Co., Sigma, St. Louis, MO, USA) or scrambled siRNA control (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) in accordance with the manufacturer’s instructions. Scramble control siRNA was referred as "control siRNA" in Figures. After transfection for 72 hours, cells were colonized by Salmonella for 30 minutes, washed, and incubated for 4 hours in DMEM with Gentamicin (500 µg/ml), and then the levels of claudin-2 and β-actin were assessed by western blot.
Real Time Quantitative PCR
Total RNA was extracted from epithelial cell monolayers or mouse colonic epithelial cells using TRIzol reagent (Invitrogen, Grand Island, NY, USA). The RNA integrity was verified by gel electrophoresis. RNA reverse transcription was done using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's directions. The RT-cDNA reaction products were subjected to quantitative real-time PCR using the MyiQ single-color real-time PCR detection system (Bio-Rad) and iQ SYBR green supermix (Bio-Rad) according to the manufacturer's directions. All expression levels were normalized to β-actin levels of the same sample. Percent expression was calculated as the ratio of the normalized value of each sample to that of the corresponding untreated control cells. All real-time PCR reactions were performed in triplicate. All PCR primers were designed using Lasergene software () (DNAStar, Madison, WI, USA).
S. typhimurium Invasion of Human Epithelial Monolayers
Infection of SKCO15 cells was performed by a previously described method 
. Bacterial solution was added, and bacterial invasion was assessed after 30 minutes. Cell-associated bacteria, representing bacteria adhered to and/or internalized into the monolayers, were released by incubation with 100 ml 1% Triton X-100 (Sigma). Internalized bacteria were those obtained from lysis of the epithelial cells with 1% Triton X-100 30 minutes after the addition of gentamicin(500 µg/ml). For both cellassociated and internalized bacteria, 0.9 ml LB broth was added, and each sample was vigorously mixed and quantitated by plating for CFU on MacConkey agar medium.
Data are expressed as mean ± SD. Differences between two samples were analyzed by Student's t test. P-values of 0.05 or less were considered statistically significant. Differences among three or more groups were analyzed using ANOVA (SAS 9.2 version, SAS Institute Inc., Cary, NC, USA).